Outcomes of Continuous Enteral Vancomycin Infusion in Intensive Care Unit Patients: A Novel Treatment Modality for Severe Clostridium Difficile Colitis.

Background and objective Severe Clostridium difficile (C. difficile) infection (CDI)-related colitis is associated with high morbidity and mortality. Current guidelines recommend oral vancomycin plus intravenous metronidazole as the first-line treatment and early total colectomy in case of medication failure. In critically ill patients at high surgical risk and with multiple comorbidities, loop ileostomy creation and enteral vancomycin infusion have been employed albeit with limited success. We hypothesized that continuous enteral vancomycin (CEV) infusion via a postpyloric feeding tube would provide a less invasive, efficacious, and safer route to treat high surgical risk patients. Methods All adult (>18 years) non-pregnant patients admitted to the ICU for severe CDI from October 2012 to October 2016 and received CEV after the failure of conventional therapy were included. Vancomycin was prepared as a 1-2-mg/ml enteral solution and run continuously through a feeding pump at 42 ml/hour via a post-pyloric feeding tube. The primary efficacy endpoint was clinical improvement defined as (a) decrease in stool output, (b) decreased vasopressor requirement, or (c) improved leukocytosis, and the secondary endpoint was treatment failure defined as the need for total colectomy or death due to severe CDI. Results Our cohort comprised 11 patients in total. The median age of the participants was 64 years, and there were more females (67%) than males (36%). Clinical improvement was seen in seven patients (63%); treatment failure documented as the need for total colectomy was observed in two patients (18%), and death attributable to CDI occurred in three patients (27%). Conclusion CEV resulted in clinical improvement in most patients with severe CDI who were at high surgical risk. Sustained intestinal vancomycin delivery may increase luminal concentration and bactericidal effect. The use of a feeding tube and pump provides an effective and less invasive route of vancomycin delivery in critically ill patients.

Limited efficacy of zonisamide in the treatment of refractory infantile spasms.

A series of relatively small studies collectively suggest that zonisamide may be effective in the treatment of infantile spasms. Using a large single-center cohort of children with infantile spasms, we set out to evaluate the efficacy and safety of zonisamide. We retrospectively identified all patients with infantile spasms who were treated with zonisamide at our center. For each patient, we recorded dates of birth, infantile spasms onset, response (if any), and most recent follow-up. To quantify zonisamide exposure, we recorded daily dosage and patient weight at each sequential encounter so as to allow calculation of peak and weighted-average weight-based dosage. We identified 87 children who were treated with zonisamide, of whom 78 had previously been treated with hormonal therapy or vigabatrin. Peak and weighted-average zonisamide dosage were 7.1 (interquartile range 3.6, 10.2) and 5.4 (interquartile range 3.0, 8.9) mg/kg/day, respectively. Whereas five (6%) patients exhibited resolution of epileptic spasms, only two (2%) patients exhibited video-EEG confirmed resolution of both epileptic spasms and hypsarrhythmia (electroclinical response). Importantly, both electroclinical responders had not previously been treated with hormonal therapy or vigabatrin; in contrast, none of the 78 children with prior failure of hormonal therapy or vigabatrin subsequently responded to zonisamide. Zonisamide was well tolerated, and there were no deaths. This study suggests that zonisamide exhibits favorable tolerability but very limited efficacy among patients who do not respond to first-line therapy.

Beta 2-microglobulin regulates amyloid precursor-like protein 2 expression and the migration of pancreatic cancer cells.

Beta 2-microglobulin (ß2m) is a component of the major histocompatibility complex (MHC) class I molecule, which presents tumor antigens to T lymphocytes to trigger cancer cell destruction. Notably, ß2m has been reported as persistently expressed, rather than down regulated, in some tumor types. For renal cell and oral squamous cell carcinomas, ß2m expression has been linked to increased migratory capabilities. The migratory ability of pancreatic cancer cells contributes to their metastatic tendencies and lethal nature. Therefore, in this study, we examined the impact of ß2m on pancreatic cancer cell migration. We found that ß2m protein is amply expressed in several human pancreatic cancer cell lines (S2-013, PANC-1, and MIA PaCa-2). Reducing ß2m expression by short interfering RNA (siRNA) transfection significantly slowed the migration of the PANC-1 and S2-013 cancer cell lines, but increased the migration of the MIA PaCa-2 cell line. The amyloid precursor-like protein 2 (APLP2) has been documented as contributing to pancreatic cancer cell migration, invasiveness, and metastasis. We have previously shown that ß2m/HLA class I/peptide complexes associate with APLP2 in S2-013 cells, and in this study we also detected their association in PANC-1 cells but not MIA PaCa-2 cells. In addition, siRNA down regulation of ß2m expression diminished the expression of APLP2 in S2-013 and PANC-1 but heightened the level of APLP2 in MIA PaCa-2 cells, consistent with our migration data and co-immunoprecipitation data. Thus, our findings indicate that ß2m regulates pancreatic cancer cell migration, and furthermore suggest that APLP2 is an intermediary in this process.

Exosomes harbor B cell targets in pancreatic adenocarcinoma and exert decoy function against complement-mediated cytotoxicity.

Although B cell response is frequently found in cancer, there is little evidence that it alters tumor development or progression. The process through which tumor-associated antigens trigger humoral response is not well delineated. We investigate the repertoire of antigens associated with humoral immune response in pancreatic ductal adenocarcinoma (PDAC) using in-depth proteomic profiling of immunoglobulin-bound proteins from PDAC patient plasmas and identify tumor antigens that induce antibody response together with exosome hallmark proteins. Additional profiling of PDAC cell-derived exosomes reveals significant overlap in their protein content with immunoglobulin-bound proteins in PDAC plasmas, and significant autoantibody reactivity is observed between PDAC cell-derived exosomes and patient plasmas compared to healthy controls. Importantly, PDAC-derived exosomes induce a dose-dependent inhibition of PDAC serum-mediated complement-dependent cytotoxicity towards cancer cells. In summary, we provide evidence that exosomes display a large repertoire of tumor antigens that induce autoantibodies and exert a decoy function against complement-mediated cytotoxicity.

The role of vascular endothelial growth factor, interleukin 8, and insulinlike growth factor in sustaining autophagic DIRAS3-induced dormant ovarian cancer xenografts.

BACKGROUND: Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells. METHODS: Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models. RESULTS: Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P < .05) and curing a percentage of mice. CONCLUSIONS: Treatment with a combination of anti-VEGF, anti-IL-8, and anti-IGF receptor antibodies prevented the outgrowth of dormant cells and prolonged survival in a preclinical model.

Targeting the Leukemia Antigen PR1 with Immunotherapy for the Treatment of Multiple Myeloma.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivoConclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386-96. ©2018 AACR.

Neuropilin-1 mediates neutrophil elastase uptake and cross-presentation in breast cancer cells.

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR.

Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome.

The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2'-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.

Amyloid precursor protein and amyloid precursor-like protein 2 in cancer.

Amyloid precursor protein (APP) and its family members amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) are type 1 transmembrane glycoproteins that are highly conserved across species. The transcriptional regulation of APP and APLP2 is similar but not identical, and the cleavage of both proteins is regulated by phosphorylation. APP has been implicated in Alzheimer's disease causation, and in addition to its importance in neurology, APP is deregulated in cancer cells. APLP2 is likewise overexpressed in cancer cells, and APLP2 and APP are linked to increased tumor cell proliferation, migration, and invasion. In this present review, we discuss the unfolding account of these APP family members' roles in cancer progression and metastasis.

The pharmacokinetic profile of methylphenidate use in pregnancy: A study in mice.

The purpose of this study was to quantify the amounts of the d- and l-threo enantiomers of methylphenidate in maternal plasma, placenta, and maternal and fetal brain tissue following prenatal exposure and to establish a pharmacokinetic profile for MPH during pregnancy. Due to increasing rates of use of methylphenidate amongst females of childbearing age, it is important to understand the extent of exposure to the fetus. Briefly, pregnant mice were injected with 5 mg/kg methylphenidate at 18 days gestation, and tissue was collected 1, 5, 10, 30, 60, and 120 min following injection. Methylphenidate was extracted from tissue via solid phase extraction, and concentrations were determined using liquid chromatography-mass spectrometry (LC-MS). Because methylphenidate is administered as a racemic mixture of d- and l-threo enantiomers and the d-enantiomer is more pharmacologically active, the enantiomers were quantified separately. Interestingly, we found that methylphenidate does cross the placenta and enter the fetal brain. Although the highest concentrations were achieved in maternal brain, the concentrations of d- and l-methylphenidate in fetal brain were comparable to those of maternal plasma. Additionally, both d- and l-methylphenidate had longer half-lives in placenta than in maternal or fetal brain. Interestingly, there was a bimodal peak in maternal brain concentrations, at 5 min and again at 60 min, which was not observed in maternal plasma. Finally, the total exposure (as represented by area under the curve) was statistically significantly higher for the active d-enantiomer than the l-enantiomer in maternal brain tissue. In conclusion, methylphenidate crosses the placenta and reaches measurable concentrations in fetal brain. Although long-term behavioral and developmental studies are needed to determine specific outcomes of prenatal exposure, discussion with pregnant patients on the potential risks of methylphenidate exposure is warranted.

Amyloid precursor-like protein 2 (APLP2) affects the actin cytoskeleton and increases pancreatic cancer growth and metastasis.

Amyloid precursor-like protein 2 (APLP2) is aberrantly expressed in pancreatic cancer. Here we showed that APLP2 is increased in pancreatic cancer metastases, particularly in metastatic lesions found in the diaphragm and intestine. Examination of matched human primary tumor-liver metastasis pairs showed that 38.1% of the patients had positive APLP2 expression in both the primary tumor and the corresponding liver metastasis. Stable knock-down of APLP2 expression (with inducible shRNA) in pancreatic cancer cells reduced the ability of these cells to migrate and invade. Loss of APLP2 decreased cortical actin and increased intracellular actin filaments in pancreatic cancer cells. Down-regulation of APLP2 decreased the weight and metastasis of orthotopically transplanted pancreatic tumors in nude mice.

APLP2 regulates the expression of MHC class I molecules on irradiated Ewing's sarcoma cells.

Ewing's sarcoma (EWS) is a pediatric cancer that is conventionally treated by surgery, chemotherapy, and radiation therapy. Innovative immunotherapies to treat EWS are currently under development. Unfortunately for EWS patients, when the disease is found to be resistant to current therapeutic approaches, the prognosis is predictably grim. Radiation therapy and immunotherapy could potentially synergize in the eradication of EWS, as some studies have previously shown that irradiation increases the presence of immune receptors, including MHC class I molecules, on the surface of tumor cells. However, EWS cells have been reported to express low levels of MHC class I molecules, a phenotype that would inhibit T-cell mediated lysis. We have previously demonstrated that the transgene-driven overexpression of amyloid ß (A4) precursor-like protein 2 (APLP2) reduces the expression of MHC class I molecules on the surface of human cervical carcinoma HeLa cells. We thus examined whether endogenously expressed APLP2 downregulates MHC class I expression on EWS cells, particularly upon irradiation. We found that irradiation induces the relocalization of APLP2 and MHC class I molecules on the surface of EWS cells, redistributing cells from subpopulations with relatively low APLP2 and high MHC class I into subpopulations with relatively high APLP2 and low MHC class I surface expression. Consistent with these findings, the transfection of an APLP2-targeting siRNA into EWS cells increased MHC class I expression on the cell surface. Furthermore, APLP2 was found by co-immunoprecipitation to bind to MHC class I molecules. Taken together, these findings suggest that APLP2 inhibits MHC class I expression on the surface of irradiated EWS cells by a mechanism that involves APLP2/MHC class I interactions. Thus, therapeutic strategies that limit APLP2 expression may boost the ability of T cells to recognize and eradicate EWS in patients.

Amyloid precursor-like protein 2 suppresses irradiation-induced apoptosis in Ewing sarcoma cells and is elevated in immune-evasive Ewing sarcoma cells.

Despite surgery, chemotherapy, and radiotherapy treatments, the children, adolescents, and young adults who are diagnosed with metastasized Ewing sarcoma face a dismal prognosis. Amyloid precursor-like protein 2 (APLP2) has recently been implicated in the survival of cancer cells and in our current study, APLP2's contribution to the survival of Ewing sarcoma cells was examined. APLP2 was readily detected in all Ewing sarcoma cell lines analyzed by western blotting, with the TC71 Ewing sarcoma cells expressing the lowest level of APLP2 among the lines. While irradiation induces apoptosis in TC71 Ewing sarcoma cells (as we determined by quantifying the proportion of cells in the sub-G 1 population), transfection of additional APLP2 into TC71 decreased irradiation-induced apoptosis. Consistent with these findings, in parallel studies, we noted that isolates of the TC71 cell line that survived co-culture with lymphokine-activated killer (LAK) cells (which kill by inducing apoptosis in target cells) displayed increased expression of APLP2, in addition to smaller sub-G 1 cell populations after irradiation. Together, these findings suggest that APLP2 lowers the sensitivity of Ewing sarcoma cells to radiotherapy-induced apoptosis and that APLP2 expression is increased in Ewing sarcoma cells able to survive exposure to cytotoxic immune cells.

Relevance of amyloid precursor-like protein 2 C-terminal fragments in pancreatic cancer cells.

In some cellular systems, particularly neurons, amyloid precursor-like protein 2 (APLP2), and its highly homologous family member amyloid precursor protein (APP), have been linked to cellular growth. APLP2 and APP undergo regulated intramembrane proteolysis to produce C-terminal fragments. In this study, we found comprehensive expression of APLP2 C-terminal fragments in a panel of pancreatic cancer cell lines; however, APP C-terminal fragments were notably limited to the BxPC3 cell line. Extensive glycosaminoglycan modification on APLP2 was also found in the majority of pancreatic cancer cell lines. Glycosaminoglycan-modified and -unmodified APLP2, and particularly APLP2 C-terminal fragments, also demonstrated increased expression in oncogene-transformed pancreatic ductal cells. Additionally, elevated APLP2 levels were confirmed in human pancreatic cancer tissue. Downregulation of APLP2 and APP expression, alone or in combination, caused a decrease in the growth of a pancreatic cancer cell line with representatively low APP C-terminal fragment expression, the S2-013 cell line. Furthermore, we found that treatment with ß-secretase inhibitors to block formation of APLP2 C-terminal fragments decreased the growth and viability of S2-013 cells, without affecting the survival of a non-transformed pancreatic ductal cell line. In conclusion, our studies demonstrate that abundant APLP2, but not APP, C-terminal fragment expression is conserved in pancreatic cancer cell lines; however, APP and APLP2 equally regulated the growth of S2-013 pancreatic cancer cells. Chiefly, our discoveries establish a role for APLP2 in the growth of pancreatic cancer cells and show that inhibitors preventing APLP2 cleavage reduce the viability of pancreatic cancer cells.

Regulation of major histocompatibility complex class I molecule expression on cancer cells by amyloid precursor-like protein 2.

The three members of the amyloid precursor protein family in mammals [amyloid precursor protein, amyloid precursor-like protein 1, and amyloid precursor-like protein 2 (APLP2)] have been implicated in a large array of intracellular processes, which include development, transcription, apoptosis, metabolism, and the cell cycle. A series of studies by our laboratories has demonstrated that APLP2 is highly expressed by many cancer cell lines (with the highest expression in pancreatic cancer cell lines) and that it facilitates major histocompatibility complex (MHC) class I molecule endocytosis. This review focuses on this recently revealed function of APLP2 relevant to tumor immunology: that it acts as a novel regulator of MHC class I molecule surface expression.